循环 RNA ZFR 通过 miR-624-3p/WEE1 轴促进肝癌细胞增殖和上皮-间充质转化过程。
Circulating RNA ZFR promotes hepatocellular carcinoma cell proliferation and epithelial-mesenchymal transition process through miR-624-3p/WEE1 axis.
机构信息
Department of General Surgery, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China.
Department of Breast Cancer, Shaanxi Provincial Cancer Hospital, Xi'an 710000, China.
出版信息
Hepatobiliary Pancreat Dis Int. 2024 Feb;23(1):52-63. doi: 10.1016/j.hbpd.2023.07.003. Epub 2023 Jul 17.
BACKGROUND
Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is the fourth leading cause of cancer-related deaths worldwide. Previous evidence shows that the expression of circulating RNA ZFR (circZFR) is upregulated in HCC tissues. However, the molecular mechanism of circZFR in HCC is unclear.
METHODS
Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was employed to detect the expression of circZFR, microRNA-624-3p (miR-624-3p) and WEE1 in HCC tissues and cells. RNase R assay and actinomycin D treatment assay were used to analyze the characteristics of circZFR. For functional analysis, the capacities of colony formation, cell proliferation, cell apoptosis, migration and invasion were assessed by colony formation assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry assay and transwell assay. Western blot was used to examine the protein levels of WEE1 and epithelial-mesenchymal transition (EMT)-related proteins. The interactions between miR-624-3p and circZFR or WEE1 were validated by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Xenograft models were established to determine the role of circZFR in vivo.
RESULTS
circZFR and WEE1 were upregulated, while miR-624-3p expression was reduced in HCC tissues and cells. circZFR could sponge miR-624-3p, and WEE1 was a downstream gene of miR-624-3p. Knockdown of circZFR significantly reduced the malignant behaviors of HCC and that co-transfection with miR-624-3p inhibitor restored this change. Overexpression of WEE1 abolished the inhibitory effect of miR-624-3p mimic on HCC cells. Mechanistically, circZFR acted as a competitive endogenous RNA (ceRNA) to regulate WEE1 expression by targeting miR-624-3p. Furthermore, in vivo studies have illustrated that circZFR knockdown inhibited tumor growth.
CONCLUSIONS
circZFR knockdown reduced HCC cell proliferation, migration and invasion and promoted apoptosis by regulating the miR-624-3p/WEE1 axis, suggesting that the circZFR/miR-624-3p/WEE1 axis might be a potential target for HCC treatment.
背景
肝细胞癌（HCC）是最常见的原发性肝癌，是全球癌症相关死亡的第四大主要原因。先前的证据表明，circZFR 在 HCC 组织中的表达上调。然而，circZFR 在 HCC 中的分子机制尚不清楚。
方法
采用定量逆转录聚合酶链反应（qRT-PCR）检测 HCC 组织和细胞中 circZFR、microRNA-624-3p（miR-624-3p）和 WEE1 的表达。采用 RNase R 分析和放线菌素 D 处理试验分析 circZFR 的特征。通过集落形成试验、5-乙炔基-2'-脱氧尿苷（EdU）试验、流式细胞术分析和 Transwell 试验评估集落形成、细胞增殖、细胞凋亡、迁移和侵袭能力。Western blot 用于检测 WEE1 和上皮间质转化（EMT）相关蛋白的蛋白水平。通过双荧光素酶报告基因检测和 RNA 免疫沉淀（RIP）试验验证 miR-624-3p 与 circZFR 或 WEE1 的相互作用。建立异种移植模型以确定 circZFR 在体内的作用。
结果
circZFR 和 WEE1 上调，而 miR-624-3p 在 HCC 组织和细胞中表达下调。circZFR 可以海绵 miR-624-3p，而 WEE1 是 miR-624-3p 的下游基因。circZFR 的敲低显著降低了 HCC 的恶性行为，而 miR-624-3p 抑制剂的共转染恢复了这种变化。WEE1 的过表达消除了 miR-624-3p 模拟物对 HCC 细胞的抑制作用。机制上，circZFR 通过靶向 miR-624-3p 作为竞争性内源 RNA（ceRNA）来调节 WEE1 表达。此外，体内研究表明，circZFR 敲低抑制肿瘤生长。
结论
circZFR 敲低通过调节 miR-624-3p/WEE1 轴减少 HCC 细胞增殖、迁移和侵袭，促进细胞凋亡，表明 circZFR/miR-624-3p/WEE1 轴可能是 HCC 治疗的潜在靶点。
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